Matrix for sustained-release preparation

ABSTRACT

The present invention relates to a matrix for sustained-release preparation comprising an ester formed at a terminal carboxyl group of a straight-chain polyester which essentially consists of an α-hydroxymonocarboxylic acid. The matrix is stable to light, heat, moisture, coloring etc., and is of low toxicity. The sustained-release preparation produced by using the ester of the present invention offers stable drug release over an extended period of time, ensuring sustained stable effect. Furthermore, the sustained-release preparation does not show excess drug release just after administration.

BACKGROUND OF THE INVENTION

The present invention relates to a matrix for sustained-releasepreparation and a sustained-release preparation comprising it.

EP-A 481732 (Japanese Patent Unexamined Publication No. 112468/1993)describes a base for sustained-release preparation comprising a mixtureof polylactic acid and a glycolic acid/hydroxycarboxylic acid [HOCH(C₂₋₈alkyl)COOH] copolymer.

Japanese Patent Unexamined Publication No. 212436/1990 describes a basefor sustained-release preparation obtained by direct dehydrativepoly-condensation process of lactic acid and/or glycolic acid and anoxycarboxylic acid.

Japanese Patent Unexamined Publication No. 173746/1992 describes asustained-release drug-polymer complex prepared by adding a drug to apolymer mixture of a lactic acid/glycolic acid copolymer andpoly-γ-butyrolactone, poly-δ-valerolactone and/or poly-ε-caprolactone.

Japanese Patent Unexamined Publication No. 212423/1987 describespolymers or copolymers of esters of hydroxypoly carboxylic acids such asan ethyl ester of polymalic acid.

Japanese Patent Unexamined Publication No. 92641/1988 describesβ-benzylmalate.lactic acid copolymer.

However, these are different in structure from the ester formed at aterminal carboxyl group of a straight-chain polyester which essentiallyconsists of an α-hydroxymonocarboxylic acid.

In sustained-release preparations wherein a drug is dispersed in abiodegradable polymer, it is desirable that drug release be controlledfreely. In general, drug release duration for a sustained-releasepreparation depends on the composition and molecular weight of the basebiodegradable polymer. Initial drug release following administration ofthe sustained-release preparation is sometimes excessive, which canresult in a rapidly increased local drug concentration, and hence arapidly increased blood level, leading to undesirable action. There istherefore need to develop a matrix for sustained-release preparationenabling production of a sustained-release preparation of low initialdrug release.

According to the present invention, there is provided:

(1) A matrix for sustained-release preparation comprising an esterformed at a terminal carboxyl group of a straight-chain polyester whichessentially consists of an α-hydroxymonocarboxylic acid, the polyesterhaving a weight-average molecular weight of about 1,500 to about 50,000,

(2) The matrix according to term (1) above, wherein the straight-chainpolyester is a lactic acid/glycolic acid copolymer,

(3) The matrix according to term (1) above, wherein the ester is analkyl ester,

(4) The matrix according to term (3) above, wherein the alkyl ester is aC₁₋₃ alkyl ester,

(5) A sustained-release preparation which comprises the matrix asdefined in term (1) above and a biologically active peptide,

(6) The sustained-release preparation according to term (5) above,wherein the biologically active peptide is an LH-RH analogue,

(7) The sustained-release preparation according to term (6) above,wherein the LH-RH analogue is an LH-RH antagonist,

(8) The sustained-release preparation according to term (5) above,wherein the biologically active peptide is a cytokine,

(9) The sustained-release preparation according to term (8) above,wherein the cytokine is an interferon,

(10) An injectable preparation which comprises the sustained-releasepreparation as defined in term (5) above,

(11) An ester formed at a terminal carboxyl group of a straight-chainpolyester which essentially consists of an α-hydroxymonocarboxylic acid,the polyester having a weight-average molecular weight of about 1,500 toabout 50,000,

(12) The ester according to term (11) above, which is an ester formed ata terminal carboxyl group of a lactic acid/glycolic acid copolymer,

(13) The ester according to term (11) above, which is an alkyl ester,and

(14) The ester according to term (13) above, which is a C₁₋₃ alkylester.

DETAILED DESCRIPTION OF THE INVENTION

In the present specification, weight-average molecular weight andnumber-average molecular weight are those in terms of polystyrene asdetermined by gel permeation chromatography (GPC). Measurements weretaken using a GPC column KF804L×2 (produced by Showa Denko) withchloroform as a mobile phase.

The dispersity is calculated by the formula: (weight-average molecularweight/number-average molecular weight).

In the present invention, the straight-chain polyester having a terminalcarboxyl group essentially consists of an α-hydroxymonocarboxylic acid,has the weight-average molecular weight of about 1,500 to about 50,000is sparingly soluble or insoluble in water, is biocompatible, and isbiodegradable.

A straight-chain polyester having a terminal carboxyl is astraight-chain polyester in which the number-average molecular weight byGPC determination is almost the same as that by end-group determination.

The number-average molecular weight is calculated as follows:

First, the polyester (about 1 to 3 g) is dissolved in a mixed solvent ofacetone (25 ml) and methanol (5 ml); the solution is quickly titratedwith a 0.05N alcoholic solution of potassium hydroxide while stirring atroom temperature (20° C.) with phenolphthalein as an indicator todetermine the carboxyl group content; the number-average molecularweight by end-group determination is calculated from the followingequation:

    Number-average molecular weight by end-group determination=20,000×A/B

where A is the weight mass (g) of the polyester, and B is the amount(ml) of the 0.05N alcoholic potassium hydroxide solution added until thetitration end point is reached.

This value is hereinafter referred to as the number-average molecularweight by end-group determination.

For example, in the case of a polymer having a terminal carboxyl groupas synthesized from one or more α-hydroxymonocarboxylic acids bycatalyst-free dehydrative poly-condensation process, the number-averagemolecular weights by GPC determination and end-group determinationalmost agree with each other. On the other hand, in the case of apolyester having substantially no free terminal carboxyl group assynthesized from a cyclic dimer by ring-opening polymerization processusing a catalyst, the number-average molecular weight by end-groupdetermination is significantly higher than that by GPC determination.This difference makes it possible to clearly differentiate a polyesterhaving a terminal carboxyl group from a polyester having substantiallyno terminal carboxyl group.

While the number-average molecular weight by end-group determination isan absolute value, that by GPC determination is a relative value thatvaries depending on various analytical conditions (e.g., kind of mobilephase, kind of column, reference substance, slice width, baseline); itis therefore difficult to have an absolute numerical representation ofboth values. However, the fact that the number-average molecular weightsby GPC determination and end-group determination almost agree with eachother means that the number-average molecular weight by end-groupdetermination falls within the range from about 0.4 to 2 times,preferably from about 0.5 to 2 times, and more preferably from about 0.8to 1.5 times, that by GPC determination. Also, the fact that thenumber-average molecular weight by end-group determination issignificantly higher than that by GPC determination means that thenumber-average molecular weight by end-group determination is over about2 times that by GPC determination.

The weight-average molecular weight of the straight-chain polyester ofthe present invention which essentially consists of anα-hydroxymonocarboxylic acid (hereinafter also referred to asstraight-chain polyester having a terminal carboxyl group) is about1,500 to 50,000. The weight-average molecular weight is preferably about2,000 to 40,000, more preferably about 5,000 to 25,000.

Reference to an α-hydroxymonocarboxylic acid includes both a singleα-hydroxymonocarboxylic acid or mixtures of severalα-hydroxymonocarboxylic acids.

Examples of the straight-chain polyester having a terminal carboxylgroup is an α-hydroxymonocarboxylic acid(s) (e.g., glycolic acid, lacticacid, 2-hydroxybutyric acid, 2-hydroxyvaleric acid,2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid,2-hydroxyisocaproic acid, 2-hydroxycaprylic acid) in the form of ahomopolymer (e.g., lactic acid polymer), a copolymer (e.g., lacticacid/glycolic acid copolymer, 2-hydroxybutyric acid/glycolic acidcopolymer) or a mixture of these homopolymers and/or copolymers (e.g.,mixture of lactic acid polymer and 2-hydroxybutyric acid/glycolic acidcopolymer).

Particularly preferable straight-chain polyesters having a terminalcarboxyl group include the lactic acid/glycolic acid copolymer describedin Japanese Patent Unexamined Publication No. 28521/1986 and the mixtureof (A) polylactic acid and (B) glycolic acid/α-hydroxycarboxylic acid[HOCH(C₂₋₈ alkyl)COOH] copolymer described in Japanese Patent UnexaminedPublication No. 112468/1993.

For example, when a lactic acid/glycolic acid copolymer is used, thecontent ratio (mol %) of lactic acid/glycolic acid is preferably 100/0to about 40/60, more preferably about 90/10 to 50/50. Here, lacticacid/glycolic acid which has the content ratio of 100/0 means ahomopolymer of lactic acid.

The weight-average molecular weight of the lactic acid/glycolic acidcopolymer is preferably about 5,000 to 25,000, more preferably about7,000 to 20,000. The dispersity of the lactic acid/glycolic acidcopolymer (weight-average molecular weight/number-average molecularweight) is preferably about 1.2 to 4.0, more preferably about 1.5 to3.5.

The decomposition/elimination rate of a lactic acid/glycolic acidcopolymer varies widely, depending on composition or molecular weight.However, drug release duration can be extended by lowering the glycolicacid ratio or increasing the molecular weight, sincedecomposition/elimination is delayed as the glycolic acid ratiodecreases. Conversely, drug release duration can be shortened byincreasing the glycolic acid ratio or decreasing the molecular weight.

For example, when a mixture of (A) polylactic acid and (B) glycolicacid/α-hydroxycarboxylic acid [HOCH(C₂₋₈ alkyl)COOH] copolymer is used,the hydroxycarboxylic acid is preferably 2-hydroxybutyric acid,2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproicacid, or the like, with greater preference given to 2-hydroxybutyricacid. Although the hydroxycarboxylic acid may be of the D-, L- orD,L-configuration, it is preferable to use a mixture of the D- andL-configurations. In such case, the ratio of the D-/L-configuration (mol%) preferably falls within the range from about 75/25 to 25/75, morepreferably from about 60/40 to 40/60, and still more preferably fromabout 55/45 to 45/55.

With respect to the glycolic acid/α-hydroxycarboxylic acid [HOCH(C₂₋₈alkyl)COOH] copolymer (hereinafter glycolic acid copolymer), it ispreferable that the content ratio of glycolic acid to hydroxycarboxylicacid is about 10 to 75 mol %, more preferably about 20 to 75 mol %. Theweight-average molecular weight of the above-described glycolic acidcopolymer is normally about 2,000 to 50,000, preferably about 3,000 to40,000, and more preferably about 8,000 to 40,000. The dispersity of theglycolic acid copolymer (weight-average molecular weight/number-averagemolecular weight) is preferably about 1.2 to 4.0, more preferably about1.5 to 3.5.

Although the above-described polylactic acid may be of the D- orL-configuration or a mixture thereof, it is preferable to use a mixtureof the D- and L-configurations. The ratio of the D-/L-configuration (mol%) preferably falls within the range from about 75/25 to 20/80, morepreferably from about 60/40 to 25/75, and still more preferably fromabout 55/45 to 25/75. The weight-average molecular weight of thepolylactic acid is preferably about 1,500 to 30,000, more preferablyabout 2,000 to 20,000, and still more preferably about 3,000 to 15,000.The dispersity of the polylactic acid (weight-average molecularweight/number-average molecular weight) is preferably about 1.2 to 4.0,more preferably about 1.5 to 3.5.

The mixing ratio of (A) polylactic acid and (B) glycolic acid copolymer[(A)/(B) (weight %)] is normally about 10/90 to 90/10, preferably about20/80 to 80/20, and more preferably about 30/70 to 70/30. If component(A) or (B) is in excess, the preparation obtained shown nothing morethan almost the same drug release pattern as obtained with component (A)or (B) alone; zero order release pattern owing to the mixed matrix isnot obtained in the latter phase of drug release. Thedecomposition/elimination rates of glycolic acid copolymer andpolylactic acid vary widely, depending on composition or molecularweight. However, drug release duration can be extended by increasing themolecular weight of the polylactic acid or the mixing ratio (A)/(B),since the decomposition/elimination rate of glycolic acid copolymer isusually higher than that of polylactic acid. Conversely, drug releaseduration can be shortened by decreasing the molecular weight ofpolylactic acid or mixing ratio (A)/(B). Drug release duration can alsobe adjusted by altering the kind and content ratio of hydroxycarboxylicacid used.

The ester formed at the terminal carboxyl group is exemplified bypharmacologically acceptable esters and include alkyl esters, arylesters, aralkyl esters.

Here, alkyl esters are exemplified by esters of alkyl groups having 1 to6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neo-pentyl, tert-pentyl, 1-ethylpropyl, n-hexyl, isohexyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and2-ethylbutyl, which alkyl groups may have 1 to 3 substituents selectedfrom halogen atoms such as chlorine, bromine and fluorine,(C₁₋₈)alkyl-carbonyl groups such as methylcarbonyl, ethylcarbonyl andbutylcarbonyl, and the nitro group.

Examples of aryl esters include esters of aryl groups having 6 to 10carbon atoms, such as phenyl and naphthyl, which aryl groups may have 1to 3 substituents selected from halogen atoms such as chlorine, bromineand fluorine, (C₁₋₆)alkyl-carbonyl groups such as methylcarbonyl,ethylcarbonyl and butylcarbonyl, and the nitro group.

Examples of aralkyl esters include esters of aralkyl groups having 7 to19 carbon atoms, such as benzyl, phenylethyl, naphthylmethyl and trityl,which aralkyl groups may have 1 to 3 substituents selected from halogenatoms such as chlorine, bromine and fluorine, (C₁₋₆)alkyl-carbonylgroups such as methylcarbonyl, ethylcarbonyl and butylcarbonyl, and thenitro group.

The ester formed at the terminal carboxyl group is preferably an alkylester. More preferable esters are esters of alkyl groups having 1 to 6carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl,neo-pentyl, tert-pentyl, 1-ethylpropyl, n-hexyl, isohexyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and2-ethylbutyl, which alkyl groups may have 1 to 3 substituents selectedfrom (C₁₋₆)alkyl-carbonyl groups such as methylcarbonyl, ethylcarbonyland butylcarbonyl, and the nitro group.

Particularly preferable esters include esters of alkyl groups having 1to 3 carbon atoms, such as methyl, ethyl, n-propyl and isopropyl.

The ester of the present invention is produced by esterifying theterminal carboxyl group of a straight-chain polyester which essentiallyconsists of an α-hydroxymonocarboxylic acid and has the weight-averagemolecular weight of about 1,500 to about 50,000 (hereinafter alsoreferred to as starting polymer). This esterification is carried out byper se known methods as follows:

(1) The starting polymer is reacted in a mixture of a diazoalkane (e.g.,diazomethane, phenyldiazomethane, diphenyldiazomethane) and a solventthat does not interfere with the reaction (e.g., ether such astetrahydrofuran or dioxane, ester such as ethyl acetate, nitrile such asacetonitrile, halogenated hydrocarbon such as dichloromethane ordichloroethane). Reaction temperature is about 0° C. to refluxingtemperature. Reaction time is about 2 minutes to 20 hours.

(2) An alkali metal salt (e.g., sodium salt, potassium salt, lithiumsalt) of the starting polymer is reacted with an activated alkyl halide(e.g., methyl iodide, benzyl bromide, p-nitro-benzyl bromide,m-phenoxybenzyl bromide, p-t-butylbenzyl bromide, pivaloyloxymethylchloride). This reaction is carried out in a solvent that does notinterfere with the reaction (e.g., amide such as dimethylformamide,dimethylacetamide or hexamethylphosphoramide, ketone such as acetone).Reaction temperature is about 0° to 60° C. Reaction time is about 2minutes to 4 hours. The reaction is not hampered even in the presence oftriethylamine etc. in the reaction mixture.

(3) The starting polymer is reacted with an alcohol, such as methanol,ethanol or benzyl alcohol. This reaction is carried out in the presenceof a carbodiimide as a condensing agent (e.g., dicyclohexylcarbodiimide,1-ethyl-3-(3-dimethylaminoisopropyl)-carbodiimide). Reaction temperatureis about 0° C. to refluxing temperature. Reaction time is about 15minutes to 18 hours. Solvents that do not interfere with the reactionare used, including halogenated hydrocarbons, such as chloroform,dichloromethane and dichloroethane.

(4) The starting polymer is reacted with an acid halide (e.g., ethylchloroformate, benzyl chloroformate); the resulting acid anhydride isreacted with an alcohol (e.g., methanol, ethanol, benzyl alcohol) underthe conditions described in term (3) above. This acid anhydride isobtained by reacting the starting polymer with an acid halide, such asan acid chloride, in a solvent that does not interfere with the reaction(e.g., ether such as tetrahydrofuran, halogenated hydrocarbon such asdichloromethane). Reaction temperature is about 25° C. to refluxingtemperature. Reaction time is about 15 minutes to 10 hours.

The ester of the present invention is used as a matrix forsustained-release preparations, such as microcapsules.

With respect to the ester of the present invention, there is almost nohydrogen bond between carboxyl groups and almost no reaction betweenbasic drug and terminal carboxyl group. Therefore, in asustained-release preparation produced by using this ester, initial drugrelease immediately following administration is suppressed, by delay ofwater permeation into the preparation and other reasons, due toincreased base hydrophobicity. And further, the matrix forsustained-release preparation comprising the ester of the presentinvention is advantageously used as a matrix for sustained-releasepreparation capable of releasing a drug over an extended period of time,because the matrix is rather slow in the rate of hydrolysis than amatrix for sustained-release preparation consisting of a straight-chainpolyester having a terminal carboxyl group.

It is preferable that, as a matrix for sustained-release preparation,the ester of the present invention be used in combination with astraight-chain polyester having a terminal carboxyl group. Here, thestraight-chain polyester having a terminal carboxyl group is identicalwith that described above. The mixing ratio by weight is normally about100/0 to 5/95, preferably about 100/0 to 30/70, and more preferablyabout 100/0 to 50/50.

Both the ester of the present invention (C) and the straight-chainpolyester having a terminal carboxyl group (D), and used in combinationtherewith, may be a copolymer or homopolymer. Also, 3 or morestraight-chain polyesters, e.g., 1 kind of (C) and 2 kinds of (D), maybe used in combination. The kind, weight-average molecular weight,dispersion value and other factors of the straight-chain polyesterhaving a terminal carboxyl group, and the kind, weight-average molecularweight and other factors of the ester of the present invention, arechosen to obtain the desired drug release duration and to satisfactorilysuppress excess initial drug release following administration.

A typical example of such combination is the combination of a lacticacid/glycolic acid copolymer having an alkyl-esterified terminalcarboxyl group (E) and a lactic acid/glycolic acid copolymer having aterminal carboxyl group (F). The ratio by weight of (E) and (F) isnormally about 100/0 to 5/95, preferably about 100/0 to 20/80, and morepreferably about 100/0 to 50/50. Components (E) and (F) may or may nothave the same lactic acid/glycolic acid ratio, and may or may not havethe same weight-average molecular weight.

Another typical example is the combination of a polylactic acid havingan alkyl-esterified terminal carboxyl group (G) and a glycolicacid/2-hydroxybutyric acid copolymer having a terminal carboxyl group(H). The ratio by weight of (G) and (H) is normally about 100/0 to 5/95,preferably about 100/0 to 20/80, and more preferably from about 100/0 to50/50. Components (G) and (H) may or may not have the sameweight-average molecular weight.

The matrix for sustained-release preparation comprising the ester of thepresent invention is prepared as a sustained-release preparation using agiven drug.

Useful drugs include, but are not limited to, biologically activepeptides, antitumor agents, antibiotics, antipyretic analgesicanti-inflammatory agents, antitussive expectorants, sedatives, musclerelaxants, antiepileptics, antiulcer agents, antidepressants,anti-allergic agents, cardiotonics, antiarrhythmic agents, vasodilators,hypotensive diuretics, antidiabetics, anticoagulants, hemostatics,antituberculosis drugs, hormones, narcotic antagonists, osteoporosisremedies and angiogenesis inhibitors.

Biologically active peptides consisting of 2 or more amino acids andhaving a molecular weight of about 200 to 80,000 are preferred.

Examples of biologically active peptides include luteinizinghormone-releasing hormone (LH-RH) and similarly acting analogs, such asthe peptide represented by the following formula [I]:

    (Pyr)Glu-R.sub.1 -Trp-Ser-R.sub.2 -R.sub.3 -R.sub.4 -Arg-Pro-R.sub.5[I]

wherein R₁ represents His, Tyr, Trp or p-NH₂ -Phe; R₂ represents Tyr orPhe; R₃ represents Gly or a D-type amino acid residue; R₄ representsLeu, Ile or Nle; R₅ represents Gly-NH-R₆ (R₆ is H or a lower alkyl groupwith or without a hydroxyl group) or NH-R₆ (R₆ has the same definitionas defined above), or a salt thereof [see U.S. Pat. Nos. 3,853,837,4,008,209 and 3,972,859, British Patent No. 1,423,083, Proceedings ofthe National Academy of Science of the United States of America, Vol.78, pp. 6509-6512 (1981)].

With respect to formula [I] above, the D-type amino acid residue for R₃is exemplified by α-D-amino acids having up to 9 carbon atoms (e.g.,D-Leu, Ile, Nle, Val, Nval, Abu, Phe, Phg, Ser, Thr, Met, Ala, Trp,α-Aibu). These amino acid residues may have a protecting group (e.g.,t-butyl, t-butoxy, t-butoxycarbonyl) as appropriate. Acid salts (e.g.,carbonate, bicarbonate, acetate, propionate) and metal complex compounds(e.g., copper complex, zinc complex) of peptide [I] can also be used asis peptide [I].

Abbreviations for amino acids, protecting groups and others in thepeptide represented by formula [I] and the following peptides are basedon abbreviations specified by the IUPAC-IUB Commission on BiochemicalNomenclature or abbreviations in common use in relevant fields. When anoptical isomer may be present in amino acid, it is of theL-configuration, unless otherwise stated.

A representative compound of formula [I] above is a peptide having Hisfor R₁, Tyr for R₂, D-Leu for R₃, Leu for R₄ and NHCH₂ --CH₃ for R₅(acetate of this peptide, commonly termed leuprorelin acetate, ishereinafter also referred to as TAP-144).

LH-RH analogs include LH-RH antagonists (see U.S. Pat. Nos. 4,086,219,4,124,577, 4,253,997 and 4,317,815).

Examples of biologically active peptides include cytokines, such aslymphokines and monokines. Examples of lymphokines include interferons(alpha, beta, gamma) and interleukins (IL-2 through IL-12). Examples ofmonokines include an interleukin (IL-1) and tumor necrosis factor (TNF).Preferable cytokines are lymphokines, with greater preference given tointerferons (alpha, beta, gamma).

Examples of biologically active peptides include insulin, somatostatin,somatostatin derivatives (see U.S. Pat. Nos. 4,087,390, 4,093,574,4,100,117 and 4,253,998), growth hormones, prolactin,adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone(MSH), thyroid hormone-releasing hormone [represented by the structuralformula (Pyr)Glu-His-ProNH₂, hereinafter also referred to as TRH] andsalts and derivatives thereof (see Japanese Patent UnexaminedPublication Nos. 121273/1975 and 116465/1977), thyroid-stimulatinghormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone(FSH), vasopressin, vasopressin derivatives [desmopressin, see FoliaEndocrinologica Japonica, Vol. 54, No. 5, pp. 676-691 (1978)], oxytocin,calcitonin, parathyroid hormone, glucagon, gastrin, secretin,pancreozymin, cholecystokinin, angiotensin, human placental lactogen,human chorionic gonadotropin (HCG), enkephalin, enkephalin derivatives(see U.S. Pat. No. 4,277,394 and European Patent Publication No. 31567),endorphin, kyotorphin, tuftsin, thymopoietin, thymosin, thymostimulin,thymic humoral factor (THF), blood thymic factor (FTS) and derivativesthereof (see U.S. Pat. No. 4,229,438), other thymic factors [Igaku noAyumi, Vol. 125, No. 10, pp. 835-843 (1983)], colony-stimulating factor(CSF), motilin, daynorphin, bombesin, neurotensin, caerulein,bradykinin, urokinase, asparaginase, kallikrein, substance P, nervegrowth factor, cell growth factor, nerve nutrition factor, bloodcoagulation factors VIII and IX, lysozyme chloride, polymixin B,colistin, gramicidin, bacitracin, erythropoietin (EPO), thrombopoietin,endothelin-antagonistic peptides (see European Patent Publication Nos.436189, 457195 and 496452, and Japanese Patent Unexamined PublicationNos. 94692/1991 and 130299/1991), fragments of these biologically activepeptides and derivatives thereof.

Examples of antitumor agents include bleomycin, methotrexate,actinomycin D, mitomycin C, binblastin sulfate, bincrystin sulfate,daunorubicin, adriamycin, neocartinostatin, cytosinearabinoside,fluorouracil, tetrahydrofuryl-5-fluorouracil, krestin, Picibanil,lentinan, levamisole, Bestatin, adimexon, glycyrrhizin, polyI:C, polyA:Uand polyICLC.

Examples of antibiotics include gentamicin, dibekacin, Kanendomycin,lividomycin, tobramycin, amikacin, fradiomycin, sisomycin, tetracyclinehydrochloride, oxytetracycline hydrochloride, rolitetracycline,doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin,cefalothin, cefaloridine, cefotiam, cefsulodin, cefmenoxime,cefmetazole, cefazolin, cefotaxime, cefoperazon, ceftizoxime,mochisalactam, thienamycin, sulfazecin and aztreonam.

Examples of antipyretic analgesic anti-inflammatory agents includessalicyclic acid, sulpyrine, flufenamic acid, diclofenac, indomethacin,morphine, pethidine hydrochloride, levorphanol tartrate and oxymorphone.

Examples of antitussive expectorants include ephedrine hydrochloride,methylephedrine hydrochloride, noscapine hydrochloride, codeinephosphate, dihydrocodeine phosphate, allocramide hydrochloride,clofedanol hydrochloride, picoperidamine hydrochloride, chloperastine,protokylol hydrochloride, isoproterenol hydrochloride, sulbutamolsulfate and terbutaline sulfate.

Examples of sedatives include chlorpromazine, prochlorperazine,trifluoperazine, atropine sulfate and methylscopolamine bromide.

Examples of muscle relaxants include pridinol methanesulfonate,tubocurarine chloride and pancuronium bromide.

Examples of antiepileptics include phenytoin, ethosuximide,acetazolamide sodium and chlordiazepoxide.

Examples of antiulcer agents include metoclopramide and histidinehydrochloride.

Examples of antidepressants include imipramine, clomipramine noxiptilineand phenerdine sulfate.

Examples of anti-allergic agents include diphenhydramine hydrochloride,chlorpheniramine maleate, tripelenamine hydrochloride, metodirazinehydrochloride, clemizole hydrochloride, diphenylpyraline hydrochlorideand methoxyphenamine hydrochloride.

Examples of cardiotonics include trans-π-oxocamphor, theophyllol,aminophylline and etilefrine hydrochloride.

Examples of antiarrhythmic agents include propranolol, alprenolol,bufetolol and oxprenolol.

Examples of vasodilators include oxyfedrine hydrochloride, diltiazem,tolazoline hydrochloride, hexobendine and bamethan sulfate.

Examples of hypotensive diuretics include hexamethonium bromide,pentolinium, mecamylamine hydrochloride, acarazine hydrochloride andclonidine.

Examples of antidiabetics include glymidine sodium, glipizide, fenforminhydrochloride, buformin hydrochloride and metformin.

Examples of anticoagulants include heparin sodium and sodium citrate.

Examples of hemolytics include thromboplastin, thrombin, menadionesodium hydrogen sulfite, acetomenaphthone, ε-aminocaproic acid,tranexamic acid, carbazochrome sodium sulfonate and adrenochromemonoaminoguanidine methanesulfonate.

Examples of antituberculosis agents include isoniazid, ethambutol andp-aminosalicylic acid.

Examples of hormones include predonizolone, predonizolone sodiumphosphate, dexamethasone sodium sulfate, betamethasone sodium phosphate,hexestrol phosphate, hexestrol acetate and methimazole.

Examples of narcotic antagonists include levallorphan tartrate,nalorphine hydrochloride and naloxone hydrochloride.

Examples of osteoporosis remedies include (sulfur-containingalkyl)aminomethylenebisphosphonic acid.

Examples of angiogenesis suppressors include angiogenesis-suppressingsteroid [see Science, Vol. 221, p. 719 (1983)], fumagillin (see EuropeanPatent Publication No. 325119) and fumagillol derivatives (see EuropeanPatent Publication Nos. 357061, 359036, 386667 and 415294).

The above-described drugs may be used as such or as salts, preferablypharmacologically acceptable salts. Such salts include salts formed withinorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid),organic acids (e.g., carbonic acid, succinic acid, acetic acid,propionic acid, trifluoroacetic acid) etc., when the drug has a basicgroup, such as the amino group. When the drug has an acidic group, suchas the carboxyl group, such salts include salts formed with inorganicbases (e.g., alkali metals such as sodium and potassium, alkaline earthmetals such as calcium and magnesium), organic bases (e.g., organicamines such as triethylamine and basic amino acids such as arginine)etc. The drug may form a metal complex compound (e.g., copper complex,zinc complex).

Since water-soluble drugs often show excess initial release, it ispreferable to use a water-soluble drug for the present invention. Thewater solubility of a drug is defined as the n-octanol oil-waterdistribution ratio. It is preferable to use a drug whose oil-waterdistribution ratio is not higher than 1, preferably not higher than 0.1.

Oil-water distribution rates can be determined by the method describedin "Butsuri Kagaku Jikkenho," by Jitsusaburo Samejima, published byShokabo, 1961. Specifically, n-octanol and a buffer of pH 5.5 (1:1 byvolume mixture) are placed in a test tube. The buffer is exemplified byS.o slashed.erensen buffer [Ergebnisse Der Physiology, 12, 393 (1912)],Clark-Lubs buffer [Journal of Bacteriology, 2 (1), 109, 191 (1917)],MacIlvaine buffer [Journal of Biological Chemistry, 49, 183, (1921)],Michaelis buffer [Die Wassers-toffionenkonzentration, p. 186 (1914)] andKolthoff buffer [Biochemische Zeitschrift, 179, 410 (1926)]. Anappropriate amount of such a drug is placed in the test tube, which isthen stoppered and immersed in a constant-temperature chamber (25° C.)with occasional vigorous shaking. When the drug appears to havedissolved in both liquid phases to reach an equilibrium, the liquidmixture is kept standing or centrifuged; a given amount is pipetted fromeach of the upper and lower layers, and analyzed for drug concentrationin each layer, to obtain the ratio of the drug concentration in then-octanol layer to that in the water layer for the oil-waterdistribution rate.

Preferable drugs are biologically active peptides, more preferably LH-RHanalogs or cytokines. Particularly preferable drugs, include LH-RHantagonists and interferons (alpha, beta, gamma).

Examples of LH-RH antagonists include peptides and salts thereof, thatare effective in treating hormone-dependent diseases, such as prostaticcancer, prostatic hypertrophy, endometriosis, uterine myoma, precociouspuberty and breast cancer, and in contraception, including the peptidesand salts thereof, that are described in U.S. Pat. No. 5,110,904, theJournal of Medicinal Chemistry, Vol. 34, pp. 2395-2402 (1991) and RecentResults in Cancer Research, Vol. 124, pp. 113-136 (1992).

More specifically, LH-RH antagonists are exemplified by the peptidesrepresented by general formula [II]: ##STR1## wherein X represents anacyl group, R₁, R₂ and R₄ independently represent an aromatic cyclicgroup; R₃ represents a D-amino acid residue or a group represented bythe formula: ##STR2## (R₃ ' represents a heterocyclic group); R₅represents a group represented by the formula --(CH₂)_(n) --R₅ ' (n is 2or 3, R₅ ' is an amino group which may be substituted), an aromaticcyclic group or an O-glycosyl group; R₆ represents a group representedby the formula --(CH₂)_(n) --R₆ ' (n is 2 or 3, R₆ ' is an amino groupwhich may be substituted); R₇ represents a D-amino acid residue or anazaglycyl group; Q represents a hydrogen atom or a lower alkyl group,and salts thereof.

With respect to general formula [II], the acyl group for X is preferablyone derived from a carboxylic acid. Said acyl group is exemplified byC₂₋₇ alkanoyl groups, C₇₋₁₅ cycloalkenoyl groups (e.g., cyclohexenoyl),C₁₋₆ alkylcarbamoyl groups (e.g., ethylcarbamoyl), 5- or 6-memberedheterocyclic carbonyl groups (e.g., piperidinocarbonyl) and carbamoylgroups; these groups may be substituted.

The acyl group is preferably a C₂₋₇ alkanoyl group which may besubstituted (e.g., acetyl, propionyl, butyryl, isobutyryl, pentanoyl,hexanoyl, heptanoyl), more preferably a C₂₋₄ alkanoyl group which may besubstituted (e.g., acetyl, propionyl, butyryl, isobutyryl). Examples ofsubstituents include C₁₋₆ alkylamino groups (e.g., methylamino,ethylamino, diethylamino, propylamino), C₁₋₃ alkanoylamino groups (e.g.,formylamino, acetylamino, propionylamino), C₇₋₁₅ cycloalkenoylaminogroups (e.g., cyclohexenoylamino), C₇₋₁₅ arylcarbonylamino groups (e.g.,benzoylamino), 5- or 6-membered heterocyclic carboxamide groups (e.g.,tetrahydrofurylcarboxamide, pyridylcarboxamide, furylcarboxamide), thehydroxyl group, carbamoyl group, formyl group, carboxyl group, and 5- or6-membered heterocyclic groups (e.g., pyridyl, morpholino). Preferablesubstituents include 5- or 6-membered heterocyclic carboxamide groups(e.g., tetrahydrofurylcarboxamide, pyridylcarboxamide,furylcarboxamide).

X is preferably a C₂₋₇ alkanoyl group which may be substituted by a 5-or 6-membered heterocyclic carboxamide group, more preferably a C₂₋₄alkanoyl group which may be substituted by a tetrahydrofurylcarboxamidegroup. Specifically, X is exemplified by acetyl and ##STR3##

The tetrahydrofuryl group in the tetrahydrofurylcarboxamidoacetyldescribed above is preferably (2S)-tetrahydrofuryl group.

The aromatic cyclic group for R₁, R₂ or R₄ is exemplified by aromaticcyclic groups having 6 to 14 carbon atoms. Such groups include phenyl,naphthyl and anthryl, with preference given to aromatic cyclic groupshaving 6 to 10 carbon atoms, such as phenyl and naphthyl. These aromaticcyclic groups may have 1 to 5, preferably 1 to 3, appropriatesubstituents at appropriate positions thereon. Such substituents includethe hydroxyl group, halogens, amino groups substituted byaminotriazolyl, and alkoxy groups, with reference given to the hydroxylgroup, halogens, and amino groups substituted by aminotriazolyl.

Here, examples of halogens include fluorine, chlorine, bromine andiodine.

The aminotriazolyl group as a substituent for the amino group isexemplified by 3-amino-1H-1,2,4-triazol-5-yl,5-amino-1H-1,3,4-triazol-2-yl, 5-amino-1H-1,2,4-triazol-3-yl,3-amino-2H-1,2,4-triazol-5-yl, 4-amino-1H-1,2,3-triazol-5-yl and4-amino-2H-1,2,3-triazol-5-yl.

The alkoxy group is preferably an alkoxy group having 1 to 6 carbonatoms (e.g., methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy).

More preferably, R₁ is a naphthyl group or halogenophenyl group. R₂ ismore preferably a halogenophenyl. R₄ is more preferably a hydroxyphenylgroup or a phenyl group substituted by aminotriazolylamino.

The D-amino acid residue for R₃ is preferably an α-D-amino acid residuehaving 3 to 12 carbon atoms. Such amino acids include leucine,isoleucine, norleucine, valine, norvaline, 2-aminobutyric acid,phenylalanine, serine, threonine, methionine, alanine, tryptophan andaminoisobutyric acid. These amino acids may have protecting groups(e.g., those in common use in relevant technical fields, such ast-butyl, t-butoxy, t-butoxycarbonyl) as appropriate.

The heterocyclic group for R₃ ' is a 5- or 6-membered heterocyclic groupwhich contains 1 or 2 hetero atoms of nitrogen or sulfur and which maybe condensed with a benzene ring. Such heterocyclic groups includethienyl, pyrrolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl,pyridyl, 3-pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl,3-benzo[b]thienyl, 3-benzo[b]-3-thienyl, indolyl, 2-indolyl, isoindolyl,1H-indazolyl, benzimidazolyl, benzothiazolyl, quinolyl and isoquinolyl.It is particularly preferable that R₃ ' be pyridyl or 3-benzo[b]thienyl.

The aromatic cyclic group for R₅ is identical with that defined for R₁,R₂ or R₄ above. This aromatic cyclic group may have 1 to 5, preferably 1to 3, appropriate substituents at appropriate positions thereon. Suchsubstituents are identical with those defined for R₁, R₂ or R₄ above.Amino groups substituted by aminotriazolyl are preferred.

The glycosyl group in the O-glycosyl group for R₅ is preferably a hexoseor derivative group thereof. Examples of hexoses include D-glucose,D-fructose, D-mannose, D-galactose and L-galactose. Such derivativesinclude deoxy sugars (e.g., L- and D-fucose, D-quinovose, L-rhamnose)and amino sugars (e.g., D-glucosamine, D-galactosamine). Deoxy sugars(e.g., L- and D-fucose, D-quinovose, L-rhamnose) are preferred, withgreater preference given to L-rhamnose.

Substituents in the amino group for R₅ ' which may be substituted areexemplified by acyl groups, carbamoyl groups, carbazoyl groups which maybe substituted by an acyl group, and amidino groups which may be mono-or di-substituted by an alkyl.

The above-described acyl group and the acyl group in the carbazoyl groupwhich may be substituted by an acyl group are exemplified by nicotinoyl,furoyl and thenoyl.

The alkyl group in the mono- or di-alkylamidino group is astraight-chain or branched alkyl group having 1 to 4 carbon atoms. Suchalkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl and tert-butyl, with preference given to the methyl group andethyl group.

Substituents in the amino group for R₆ ' which may be substitutedinclude alkyl groups, and amidino groups which may be mono- ordi-substituted by an alkyl.

The above-described alkyl group and the alkyl group in the mono- ordi-alkylamidino group are identical with the alkyl groups defined for R₅' above.

The D-amino acid residue for R₇, preferably a D-amino acid residuehaving 3 to 9 carbon atoms, is exemplified by D-alanyl, D-leucyl,D-valyl, D-isoleucyl and D-phenylalanyl. D-amino acid residues having 3to 6 carbon atoms, such as D-alanyl and D-valyl, are more preferable.

Still more preferably, R₇ is D-alanyl.

The lower alkyl group for Q is identical with the alkyl group definedfor R₅ ' above. Preferably, Q is the methyl group. ##STR4##

When peptide [II] has one or more kinds of asymmetric carbon atoms, twoor more optical isomers are present. Such optical isomers and mixturesthereof are also included in the scope of the present invention.

Peptides represented by general formula [II] can be produced by per seknown methods. Example production of such peptides is described in U.S.Pat. No. 5,110,904 and other publications.

Peptide [II] may be used as a salt, preferably a pharmacologicallyacceptable salt. Such salts include salts formed with inorganic acids(e.g., hydrochloric acid, sulfuric acid, nitric acid), organic acids(e.g., carbonic acid, bicarbonic acid, succinic acid, acetic acid,propionic acid, trifluoroacetic acid) etc., when the peptide has a basicgroup, such as an amino group. When the peptide has an acidic group,such as a carboxyl group, such salts include salts formed with inorganicbases (e.g., alkali metals such as sodium and potassium, and alkalineearth metals such as calcium and magnesium), organic bases (e.g.,organic amines such as triethylamine, and basic amino acids such asarginine) etc. The peptide may form a metal complex compound (e.g.,copper complex, zinc complex).

Preferably, the salt of peptide [II] is a salt formed with an organicacid (e.g., carbonic acid, bicarbonic acid, succinic acid, acetic acid,propionic acid, trifluoroacetic acid), with greater preference given toa salt formed with acetic acid.

Examples of particularly preferable peptide [II] and salts thereof aregiven below.

(1)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂ orits acetate

(2)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH₂or its acetate

(3)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂or its acetate ##STR5## (5)NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg(Et₂)-Leu-hArg(Et₂)-Pro-DAlaNH₂ orits acetate

Abbreviations used in the present specification are defined as follows:

    ______________________________________                                        MAcD2Nal      N-acetyl-D-3-(2-naphthyl)alanyl                                 D4ClPhe       D-3-(4-chlorophenyl)alanyl                                      D3Pal         D-3-(3-pyridyl)alanyl                                           NMeTyr        N-methyltyrosyl                                                 DLys(Nic)     D-(epsilon-N-nicotinoyl)lysyl                                   Lys(Nisp)     (epsilon-N-isopropyl)lysyl                                      DLys(AzaglyNic)                                                                             D-[1-aza-(N-nicotinoyl)glycyl]lysyl                             DLys(AzaglyFur)                                                                             D-[1-aza-(N-2-furoyl)glycyl]lysyl                               DhArg(Et.sub.2)                                                                             D-(N,N'-diethyl)homoarginyl                                     ______________________________________                                    

With respect to the sustained-release preparation of the presentinvention, the drug content ratio, although varying depending on thekind of the drug, desired pharmacologic effect, duration of effectiveperiod and other factors, is preferably about 0.01 to 50% (w/w), morepreferably about 0.1 to 40% (w/w), and more preferably about 1 to 30%(w/w), relative to the base ester.

The sustained-release preparation of the present invention, in the formof microcapsules, for instance, can be produced by the following methodA or B, or a modification thereof.

(Method A)

First, a drug is dissolved or dispersed in water, with a drug retainingsubstance, such as gelatin, agar, alginic acid, polyvinyl alcohol or abasic amino acid, dissolved or suspended when necessary, to yield aninternal aqueous phase.

The internal aqueous phase may be supplemented with a pH regulator forretaining drug stability or solubility, such as carbonic acid, aceticacid, oxalic acid, citric acid, tartaric acid, succinic acid, phosphoricacid, sodium or potassium salt thereof, hydrochloric acid, sodiumhydroxide, arginine, lysine or salt thereof. In addition, albumin,gelatin, citric acid, sodium ethylenediaminetetraacetate, dextrin,sodium hydrogen sulfite, polyol compounds such as polyethylene glycol,etc., as drug stabilizers, and p-oxybenzoates (e.g., methyl paraben,propyl paraben), benzyl alcohol, chlorobutanol, thimerosal etc., aspreservatives, may be added.

The internal aqueous phase thus obtained is added to an ester-containingsolution (oil phase), followed by emulsification, to yield a W/Oemulsion.

The above-described ester-containing solution is prepared by dissolvingan ester in an organic solvent. Any organic solvent serves this purpose,as long as it has a boiling point not higher than about 120° C., issparingly miscible with water and dissolves the ester. Such solventsinclude halogenated hydrocarbons (e.g., dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride), fatty acid esters(e.g., butyl acetate), ethers (e.g., isopropyl ether) and aromatichydrocarbons (e.g., benzene, toluene, xylene). These solvents may beused in combination.

Emulsification is achieved by a conventional dispersing method. Usefuldispersing methods include the intermittent shaking method, the methodusing a mixer, such as a propeller mixer or a turbine mixer, thecolloidal mill method, the homogenizer method and the ultrasonicationmethod.

Next, the thus-obtained W/O emulsion is subjected to a microcapsulationprocess. Useful microcapsulation methods include the in-water dryingmethod, phase separation method and spray drying method described below,and modifications thereof.

(1) In-water drying method

After the W/O emulsion is added to another aqueous phase (third phase)to yield a W/O/W emulsion, the solvent is removed from the oil phase, toyield microcapsules.

An emulsifier may be added to the third, aqueous phase. The emulsifiermay be any one, as long as it is capable of forming a stable O/Wemulsion. Such emulsifiers include anionic surfactants (e.g., sodiumoleate, sodium stearate, sodium lauryl sulfate), nonionic surfactants[e.g., polyoxyethylene sorbitan fatty acid esters (Tween 80, Tween 60,Atlas Powder Company), polyoxyethylene castor oil derivatives (e.g.,HCO-60, HCO-50, Nikko Chemicals)], polyvinylpyrrolidone, polyvinylalcohol, carboxymethyl cellulose, lecithin, gelatin and hyaluronic acid.These emulsifiers may be used singly or in combination. Theirconcentration can be chosen as appropriate over the range from about0.001 to 20% (w/w), preferably from about 0.01 to 10% (w/w), and morepreferably from about 0.05 to 5% (w/w).

Solvent removal from the oil phase can be achieved by per se knownmethods, including the method in which the solvent is evaporated undernormal or gradually reduced pressure during stirring using a propellerstirrer, magnetic stirrer or the like, and the method in which thesolvent is evaporated while the degree of vacuum is adjusted using arotary evaporator or the like.

The thus-obtained microcapsules are centrifuged or filtered to separatethem, after which they are washed with distilled water several times toremove the free drug, drug retaining substance, emulsifier etc. adheringto the microcapsule surface. The microcapsules are then again dispersedin distilled water etc. and lyophilized. To prevent mutual aggregationof particles during washing, an antiaggregation agent may be added tothe distilled water for washing. The antiaggregation agent isexemplified by water-soluble polysaccharides such as mannitol, lactol,glucose and starches (e.g., corn starch), amino acids such as glycineand alanine, proteins such as gelatin, fibrin and collagen, andinorganic salts such as sodium chloride, sodium bromide, potassiumcarbonate and sodium hydrogen phosphate. Where necessary, this isfollowed by heating under reduced pressure to remove the water andorganic solvent from the microcapsules.

(2) Phase separation method

For producing microcapsules by the phase separation method, acoacervating agent is gradually added to the above-described W/Oemulsion, while the emulsion is stirred, to precipitate and solidify theester.

Any coacervating agent can be used, as long as it is a polymeric,mineral oil or vegetable oil compound miscible with the solvent for theester and which does not dissolve the ester. Such coacervating agentsinclude silicon oil, sesame oil, soybean oil, corn oil, cotton seed oil,coconut oil, linseed oil, mineral oil, n-hexane and n-heptane. These maybe used in combination of two or more kinds.

The thus-obtained microcapsules are filtered to separate them, afterwhich they are repeatedly washed with heptane etc. to remove thecoacervating agent. The free drug and solvent are then removed in thesame manner as in the in-water drying method.

(3) Spray drying method

The W/O emulsion is sprayed via a nozzle into the drying chamber of aspray drier to volatilize the organic solvent in the fine droplets in avery short time, to yield fine microcapsules. The nozzle is exemplifiedby the double-fluid nozzle, pressure nozzle and rotary disc nozzle. Toprevent microcapsule aggregation where desired, an aqueous solution ofthe above-described antiaggregation agent may be effectively sprayed viaanother nozzle, while the organic solvent solution containing the drugand ester is sprayed.

The microcapsules thus obtained may have the water and organic solventremoved at increased temperature under reduced pressure when necessary.

(Method B)

The sustained-release preparation of the present invention can also beproduced by dissolving or dispersing a drug and ester in a solventsubstantially immiscible with water, and then removing the solvent.

The solvent substantially immiscible with water may be any one, as longas it is substantially immiscible with water, it dissolves the ester andthe resulting polymer solution dissolves the drug. Preferably, thesolvent has a water solubility not higher than 3% at normal temperature(20° C.), and a boiling point not higher than 120° C. Such solventsinclude halogenated hydrocarbons (e.g., dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride), alkyl ethershaving 3 or more carbon atoms (e.g., isopropyl ether), fatty acid alkyl(4 or more carbon atoms) esters (e.g., butyl acetate) and aromatichydrocarbons (e.g., benzene, toluene, xylene). These may be used incombination in appropriate ratios. More preferably, the solvent is ahalogenated hydrocarbon (e.g., dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride), with greaterpreference given to dichloromethane.

Solvent removal can be achieved by per se known methods, including themethod in which the solvent is evaporated under atmospheric or graduallyreduced pressure during stirring using a propeller stirrer, magneticstirrer or the like, and the method in which the solvent is evaporatedwhile the degree of vacuum is adjusted using a rotary evaporator or thelike.

The drug is added to the ester solution in the organic solvent toachieve the drug content ratio by weight defined above, to yield anorganic solvent solution of the drug and ester. The ester concentrationin the organic solvent solution is normally about 0.01 to 80% (w/w),preferably about 0.1 to 70% (w/w), and more preferably about 1 to 60%(w/w), depending on the molecular weight of the ester and the kind oforganic solvent. The thus-obtained organic solvent solution of the drugand ester is subjected to a microcapsulation process in the same manneras for the above-described W/O emulsion. Microcapsulation is carried outby, for example, the in-water drying method, phase separation method andspray drying method, or a modification thereof, as described above.

The thus-obtained microcapsules can be administered, as such or in theform of various dosage forms of non-oral preparations (e.g.,intramuscular, subcutaneous or visceral injections or indwellablepreparations, nasal, rectal or uterine transmucosal preparations) ororal preparations (e.g., capsules such as hard capsules and softcapsules), or solid preparations such as granules and powders or liquidpreparations such as syrups, emulsions and suspensions. Thesepreparations can be produced by per se known methods in common use forpharmaceutical production.

An injectable preparation can be prepared by, for example, suspendingmicrocapsules in water, along with a dispersing agent (e.g., Tween 80,HCO-60, carboxymethyl cellulose, sodium alginate), a preservative (e.g.,methyl paraben, propyl paraben, benzyl alcohol), an isotonizing agent(e.g., sodium chloride, glycerol, mannitol, sorbitol, glucose) etc., toyield an aqueous suspension, or by dispersing it in a vegetable oil suchas olive oil, sesame oil, peanut oil, cotton seed oil or corn oil,propylene glycol, or the like, to yield an oily suspension. A morestable sustained-release injectable preparation can be produced byadding to such an injectable preparation an excipient (e.g., mannitol,sorbitol, lactose, glucose), re-dispersing the microcapsules, thenlyophilizing or spray drying the dispersion to solidify it, and addingdistilled water for injection or an appropriate dispersant at the timeof use.

When microcapsules are used as an injectable suspension, for instance,their particle size is chosen over the range from about 1 to 300 μm, aslong as the requirements concerning the degree of dispersion and needlepassage are met. Preferable, the particle size is about 5 to 150 μm.

Methods of preparing microcapsules as a sterile preparation include, butare not limited to, the method in which the entire production process issterile, the method in which gamma rays are used as sterilant, and themethod in which an antiseptic is added.

An oral preparation can be produced by, for example, adding an excipient(e.g., lactose, sucrose, starch), a disintegrating agent (e.g., starch,calcium carbonate), a binder (e.g., starch, gum arabic, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl cellulose) or a lubricant(e.g., talc, magnesium stearate, polyethylene glycol 6000) tomicrocapsules, subjecting the mixture to compressive shaping, followedby coating to mask the taste or conferring an enteric orsustained-release property when necessary. This coating can be achievedby a per se known method. Useful coating agents includehydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl cellulose,hydroxypropyl cellulose, polyoxyethylene glycol, Tween 80, PrullonicF68, cellulose acetate phthalate, hydroxypropylmethyl cellulosephthalate, hydroxypropylmethyl cellulose acetate succinate, Eudragit(Rohm Company, Germany, methacrylic acid-acrylic acid copolymer), anddyes such as titanium oxide and iron oxide red.

The nasal preparation may be solid, semi-solid or liquid. For example, asolid nasal preparation can be produced by powdering the microcapsules,as such or in mixture with an excipient (e.g., glucose, mannitol,starch, microcrystalline cellulose), a thickening agent (e.g., naturalrubbers, cellulose derivative, acrylic acid polymer) etc. A liquid nasalpreparation can be produced as an oily or aqueous suspension, in almostthe same manner as for an injectable preparation. The semi-solid nasalpreparation is preferably an aqueous or oily gel or ointment. All thesepreparations may contain a pH regulator (e.g., carbonic acid, phosphoricacid, citric acid, hydrochloric acid, sodium hydroxide), an antiseptic(e.g., p-oxybenzoate ester, chlorobutanol, benzalkonium chloride) etc.

The suppository may be an oily or aqueous solid, semi-solid or liquid.Any oily base can be used to produce a suppository, as long as it doesnot dissolve fine particle preparations. Such oily bases includeglycerides of higher fatty acids [e.g., cacao fat, uitepsols (producedby Dynamite Nobel Company)], moderate fatty acids [e.g., mygliols(produced by Dynamite Nobel Company)], and vegetable oils (e.g., sesameoil, soybean oil, cottonseed oil). Aqueous bases include polyethyleneglycols and propylene glycol. Aqueous gel bases include natural rubbers,cellulose derivatives, vinyl polymers and acrylic acid polymers.

In addition to the above-described microcapsules, the sustained-releasepreparation of the present invention can be produced by dissolving abiodegradable polymer composition containing a drug dispersed therein byan appropriate method and forming the solution into balls, rods,needles, pellets, films and other forms. The biodegradable polymercomposition is produced in accordance with, for example, the methoddescribed in Japanese Patent Examined Publication No. 17525/1975. Morespecifically, the biodegradable polymer composition can be produced bydissolving a drug and a high molecular polymer in a solvent and thenremoving the solvent by an appropriate method (e.g., spray drying, flushevaporation).

The sustained-release preparation of the present invention can beadministered intramuscularly, subcutaneously or intraviscerally as aninjectable preparation or implant, intranasally, rectally or uterinelyas transmucosal preparation, or orally [e.g., solid preparations such asa capsule (e.g., hard capsule, soft capsule), granules and powders, andliquid preparations such as syrup, emulsion and suspension]. Thesustained-release preparation of the present invention is preferablyused as an injectable preparation.

The sustained-release preparation of the present invention has a lowtoxic potential and can be used safely in mammals (e.g., humans,bovines, swines, dogs, cats, mice, rats, rabbits).

Although varying widely depending on kind, content and dosage form, andduration of release of the drug, target disease, subject animal speciesand other factors, the dose of the sustained-release preparation may beset at any level, as long as the desired effect of the drug is obtained.The dose of the drug per administration can be chosen as appropriateover the range from about 0.01 to 100 mg/kg body weight, preferably fromabout 0.05 to 50 mg/kg body weight, and more preferably from about 0.1to 10 mg/kg body weight per adult in the case of a 1-month releasepreparation.

The dose of the sustained-release preparation per administration can bechosen as appropriate within the range from about 0.1 to 500 mg/kg bodyweight, preferably from about 0.2 to 300 mg/kg body weight per adult.The frequency of administration can be chosen as appropriate, dependingon kind, content and dosage form, duration of release of the drug,target disease, subject animal species and other factors, e.g., onceevery several weeks, one every month or once every several months.

The present invention is hereinafter described in more detail by meansof the following reference examples, examples and experimental examples,which are not to be construed as limitative. In the examples below, %values are by weight, unless otherwise stated.

REFERENCE EXAMPLE 1

To a 1,000 ml four-necked flask equipped with a nitrogen inlet pipe anda cooling tube, 300 g of a 90% aqueous solution of D,L-lactic acid and100 g of a 90% aqueous solution of L-lactic acid were charged, followedby heating under reduced pressure in a nitrogen stream from 100° C. and500 mmHg to 150° C. and 30 mmHg over a 4-hour period to distill offwater. After further heating under reduced pressured at 3 to 5 mmHg and150° to 180° C. for 10 hours, the residue was cooled to yieldamber-colored polylactic acid.

The resulting polymer was dissolved in 1,000 ml of dichloromethane; thesolution was added to 60° C. hot water while stirring at constant rate.The separating pasty high molecular polymer was collected and dried at30° C. under vacuum.

The weight-average molecular and number-average molecular weights by GPCdetermination and the number-average molecular weight by end-groupdetermination of the polylactic acid thus obtained were determined to be4,200, 2,192 and 1,572, respectively; the polylactic acid was identifiedas a polyester having a terminal carboxyl group.

REFERENCE EXAMPLE 2

To a 1,000 ml four-necked flask equipped with a nitrogen inlet pipe anda cooling tube, 182.5 g of glycolic acid and 166.6 g ofD,L-2-hydroxybutyric acid were charged, followed by heating underreduced pressure in a nitrogen stream from 100° C. and 500 mmHg to 150°C. and 30 mmHg over a 3.5-hour period to distill off water. Afterfurther heating under reduced pressured at 5 to 7 mmHg and 150° to 180°C. for 32 hours, the residue was cooled to yield an amber-coloredglycolic acid-2-hydroxybutyric acid copolymer.

The resulting polymer was dissolved in 1,000 ml of dichloromethane; thesolution was added to 60° C. hot water while stirring at constant rate.The separating pasty high molecular polymer was collected and dried at25° C. under vacuum.

The weight-average molecular and number-average molecular weights by GPCdetermination and the number-average molecular weight by end-groupdetermination of the glycolic acid-2-hydroxybutyric acid copolymer thusobtained were determined to be 14,700, 5,700 and 2,400, respectively;the copolymer was identified as a polyester having a terminal carboxylgroup.

EXAMPLE 1

To a mixture of 168 ml of a 40% aqueous solution of potassium hydroxideand 824 ml of ethyl ether, 81.5 g of nitrosomethylurea was added littleby little, while the mixture was stirred under ice cooling. Theresulting yellow ether layer was separated, and dried with granularpotassium hydroxide, followed by removal of potassium hydroxide, toyield about 800 ml of a diazomethane solution.

80 g of polylactic acid having a weight-average molecular weight ofabout 5,000, produced in the same manner as in Reference Example 1, wasdissolved in 500 ml of dichloromethane; this solution was stirred andcooled. While the solution was ice cooled, the above-describeddiazomethane solution was added dropwise, followed by stirring at roomtemperature for 2 hours. After the solution was kept standing overnight,the solvent was distilled off under reduced pressure; the residue wasvacuum dried at room temperature to yield 79 g of the methyl ester ofpolylactic acid.

The weight-average and number-average molecular weights by GPCdetermination and the number-average molecular weight by end-groupdetermination of the polylactic acid methyl ester thus obtained weredetermined to be 5,250, 2,960 and 1,820, respectively, the residualcarboxyl group content as lactic acid, by end-group determination, beingunder 0.1%; the ester was identified as a polyester having no terminalcarboxyl group.

EXAMPLE 2

To a mixture of 168 ml of a 40% aqueous solution of potassium hydroxideand 1,000 ml of ethyl ether, 104 g of nitrosoethylurea was added littleby little, while the mixture was stirred under ice cooling. Theresulting yellow ether layer was separated, and dried with granularpotassium hydroxide, followed by potassium hydroxide removal, to yieldabout 900 ml of a diazoethane solution.

130 g of lactic acid/glycolic acid copolymer having a weight-averagemolecular weight of about 5,000, (lactic acid/glycolic acid=50/50 (mol%)) was dissolved in 1,900 ml of dichloromethane; this solution wasstirred and cooled. While the solution was ice cooled, theabove-described diazoethane solution was added dropwise, followed bystirring at room temperature for 2 hours. After the solution was keptstanding overnight, the solvent was distilled off under reducedpressure; the residue was vacuum dried at room temperature to yield 131g of the ethyl ester of lactic acid/glycolic acid.

The weight-average and number-average molecular weights by GPCdetermination of the lactic acid/glycolic acid ethyl ester thus obtainedwere determined to be 5,120 and 2,320, respectively, the residualcarboxyl group content as lactic acid, by end-group determination, beingunder 0.1%; the ester was identified as a polyester having no terminalcarboxyl group.

EXAMPLE 3

15 g of a lactic acid/glycolic acid copolymer having a weight-averagemolecular weight of about 7,500 (lactic acid/glycolic acid=75/25 (mol%)) (Wako Pure Chemical Industries, Ltd.) and a 7.8 g of ethyl iodidewere dissolved in 150 ml of acetone. To thus obtained solution was added1.38 g of potassium carbonate and then the resulting mixture wasrefluxed for 6 hours. After the resultant solution was cooled, inorganicsubstances were removed by filtration and then the filtrate wasconcentrated under reduced pressure. The concentrate was dissolved in100 ml of dichloromethane, washed 3 times with 100 ml of 10%ethanol-water, and then dried with magnesium sulfate. After magnesiumsulfate was separated by filtration, the mixture was concentrated todryness under reduced pressure to yield 12.5 g of the ethyl ester oflactic acid/glycolic acid copolymer.

The weight-average and number-average molecular weights by GPCdetermination of the ethyl ester thus obtained were determined to be5,330 and 3,220 respectively, the residual carboxyl group content aslactic acid, by end-group determination, being under 0.1%; the ester wasidentified as a polymer having no terminal carboxyl group.

EXAMPLE 4

9 g of a lactic acid/glycolic acid copolymer having a weight-averagemolecular weight of about 7,500 (lactic acid/glycolic acid=75/25 (mol%)) (Wako Pure Chemical Industries, Ltd.) was dissolved in a mixedsolvent consisting of 20 ml of dichloromethane and 20 ml of ethanol.While thus obtained solution was cooled and stirred, 0.45 ml of triethylamine, 0.29 ml of ethyl chloroformate and 0.36 g ofN,N-dimethylaminopyridine were added. After the resulting mixture wasstirred for another 2 hours, 50 ml of dichloromethane and 50 ml of waterwere added and then dichloromethane phase was separated. Thedichloromethane phase was washed 2 times with 50 ml of 10%ethanol-water, and then dried with magnesium sulfate. After magnesiumsulfate was separated by filtration, the mixture was concentrated todryness under reduced pressure to yield 7.7 g of the ethyl ester oflactic acid/glycolic acid copolymer.

The weight-average and number-average molecular weights by GPCdetermination of the ethyl ester thus obtained were determined to be9,220 and 5,230 respectively, the residual carboxyl group content aslactic acid, by end-group determination, being under 0.1%; the ester wasidentified as a polymer having no terminal carboxyl group.

EXAMPLE 5

6 g of a lactic acid/glycolic acid copolymer having a weight-averagemolecular weight of about 7,500 (lactic acid/glycolic acid=75/25 (mol%)) (Wako Pure Chemical Industries, Ltd.) was dissolved in a mixedsolvent consisting of 60 ml of dichloromethane and 60 ml of ethanol.While thus obtained solution was cooled and stirred, 3.83 g of1-ethyl-3-(3-dimethylaminoisopropyl)carbodiimide hydrochloride was addedand then the reaction mixture was stirred overnight. After 50 ml ofwater was added, dichloromethane phase was separated. Thedichloromethane phase was washed 2 times with 40 ml of 10% ethanol-waterand dried with magnesium sulfate. After magnesium sulfate was separatedby filtration, the mixture was concentrated to dryness under reducedpressure to yield 5.2 g of the ethyl ester of lactic acid/glycolic acidcopolymer.

The weight-average and number-average molecular weights by GPCdetermination of the ethyl ester thus obtained were determined to be6,620 and 3,350 respectively, the residual carboxyl group content aslactic acid, by end-group determination, being under 0.1%; the ester wasidentified as a polymer having no terminal carboxyl group.

EXAMPLE 6

4.4 g of a 1:1 mixture of the glycolic acid/2-hydroxybutyric acidcopolymer obtained in Reference Example 2 and the polylactic acid methylester obtained in Example 1 was dissolved in 9.1 g (7.0 ml) ofdichloromethane. In this solution was dissolved 600 mg of the acetate ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂(produced by TAP Company, hereinafter referred to as biologically activepeptide A), produced by the method described in U.S. Pat. No. 5,110,904.The resulting solution was poured into 1,000 ml of a 0.1% aqueoussolution of polyvinyl alcohol (EG-40, produced by The Nippon SyntheticChemical Industry Co., Ltd.), previously adjusted to 17° C., and wasthen prepared as an O/W emulsion using a turbine type homomixer at 7,000rpm. This O/W emulsion was stirred at room temperature for 3 hours tovolatilize off the dichloromethane and solidify the oil phase, which wasthen collected via centrifugation at 1,500 rpm using a centrifuge(O5PR-22, Hitachi Limited). The oil phase was again dispersed indistilled water, followed by centrifugation to wash off the free drugetc. After the collected microcapsules were again dispersed in a smallamount of distilled water, 0.3 g of D-mannitol was added; the resultingdispersion was lyophilized to yield a powder. The biologically activepeptide A content of the microcapsules was 9.3%.

EXAMPLE 7

1.0 g of a 1:1 mixture of a lactic acid/glycolic acid copolymer having aweight-average molecular weight of 5,100 (lactic acid/glycolicacid=50/50 (mol %)) (Wako Pure Chemical Industries, Ltd.) and the lacticacid/glycolic acid ethyl ester obtained in Example 2 was dissolved in2.0 g (1.5 ml) of dichloromethane. In this solution, 40 mg of humaninterferon alpha (7.0×10⁷ IU/mg) was dispersed. The resulting dispersionwas poured into 300 ml of a 0.1% aqueous solution of polyvinyl alcohol(EG-40, produced by The Nippon Synthetic Chemical Industry Co., Ltd.),previously adjusted to 17° C., and was then prepared as an O/W emulsionusing a turbine type homomixer at 6,500 rpm. This O/W emulsion wasstirred at room temperature for 3 hours to volatilize off thedichloromethane and solidify the oil phase, which was then collected viacentrifugation using a centrifuge (O5PR-22, Hitachi Limited) at 1,500rpm. The oil phase was again dispersed in distilled water, followed bycentrifugation to wash off the free drug etc. After the collectedmicrocapsules were again dispersed in a small amount of distilled water,50 mg of D-mannitol was added; the resulting dispersion was lyophilizedto yield a powder. The human interferon alpha activity of themicrocapsules was 5.75×10⁵ IU/mg microcapsule.

EXAMPLE 8

1.0 g of the lactic acid/glycolic acid ethyl ester obtained in Example 2was dissolved in 2.0 g (1.5 ml) of dichloromethane. In this solution, 40mg of human interferon alpha (2.0×10⁸ IU/mg) was dispersed. Theresulting dispersion was poured into 300 ml of a 0.1% aqueous solutionof polyvinyl alcohol (EG-40, produced by The Nippon Synthetic ChemicalIndustry Co., Ltd.), previously adjusted to 17° C., and was thenprepared as an O/W emulsion, using a turbine type homomixer at 6,500rpm. This O/W emulsion was stirred at room temperature for 3 hours tovolatilize off the dichloromethane and solidify the oil phase, which wasthen collected via centrifugation at 1,500 rpm using a centrifuge(O5PR-22, Hitachi Limited). The oil phase was again dispersed indistilled water, followed by centrifugation to wash off the free drugetc. After the collected microcapsules were again dispersed in a smallamount of distilled water, 50 mg of D-mannitol was added; the resultingdispersion was lyophilized to yield a powder. The human interferon alphaactivity of the microcapsules was 2.48×10⁶ IU/mg microcapsule.

EXAMPLE 9

0.9 g of a 1:1 mixture of a lactic acid/glycolic acid copolymer having aweight-average molecular weight of 5,100 (lactic acid/glycolicacid=50/50 (mol %)) (Wako Pure Chemical Industries, Ltd.) and the lacticacid/glycolic acid ethyl ester obtained in Example 2 was dissolved in2.0 g (1.5 ml) of dichloromethane. In this solution 100 mg ofrecombinant insulin (Wako Pure Chemical Industries, Ltd.) was dispersed.The resulting dispersion was poured into 350 ml of a 0.1% aqueoussolution of polyvinyl alcohol (EG-40, produced by The Nippon SyntheticChemical Industry Co., Ltd.) containing 5% mannitol, previously adjustedto 18° C., and was then prepared as an O/W emulsion using a turbine typehomomixer at 6,500 rpm. This O/W emulsion was stirred at roomtemperature for 3 hours to volatilize off the dichloromethane andsolidify the oil phase, which was then collected via centrifugationusing a centrifuge (O5PR-22, Hitachi Limited) at 1,500 rpm. The oilphase was again dispersed in distilled water, followed by centrifugationto wash off the free drug etc. After the collected microcapsules wereagain dispersed in a small amount of distilled water, 50 mg ofD-mannitol was added; the resulting dispersion was lyophilized to yielda power (476 mg). The insulin content of the micro capsules was 7.95%.

EXAMPLE 10

4.5 g of a 1:1 mixture of the lactic acid/glycolic acid copolymer havinga weight-average molecular weight of 5,000 (lactic acid/glycolicacid=50/50 (mol %)) (Wako Pure Chemical Industries, Ltd.), and thelactic acid/glycolic acid ethyl ester obtained in Example 2 wasdissolved in 6.5 g (5.0 ml) of dichloromethane. To this solution wasadded 500 mg of the acetate ofN-(s)-Tetrahydrofur-2-oyl-Gly-D2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂(produced by TAP Company, hereinafter referred to as biologically activepeptide B) dissolved in 0.6 ml of distilled water, followed by mixingfor 60 seconds with a turbine type homomixer to yield a W/O emulsion.This W/O was cooled to 16° C., poured into 1,000 ml of a 0.1% aqueoussolution of polyvinyl alcohol (EG-40, produced by The Nippon SyntheticChemical Industry Co., Ltd.), previously adjusted to 16° C., and wasthen prepared as a W/O/W emulsion using a turbine type homomixer at7,000 rpm. This W/O/W emulsion was stirred at room temperature for 3hours to volatilize off the dichloromethane and solidify the W/Oemulsion, which was then collected via centrifugation at 2,000 rpm usinga centrifuge (O5PR-22, Hitachi Limited). And then, microcapsules wereobtained as a powder in the same manner as Example 6. The biologicallyactive peptide B content of the microcapsules was 9.2%.

COMPARATIVE EXAMPLE 1

1.5 g of the lactic acid/glycolic acid copolymer having a weight-averagemolecular weight of 5,100 (lactic acid/glycolic acid=50/50 (mol %))(Wako Pure Chemical Industries, Ltd.) was dissolved in 2.6 g (2.0 ml) ofdichloromethane. In this solution, 60 mg of human interferon alpha(1.5×10⁸ IU/mg) was dispersed. The resulting dispersion was poured into300 ml of a 0.1% aqueous solution of polyvinyl alcohol (EG-40, producedby The Nippon Synthetic Chemical Industry Co., Ltd.), previouslyadjusted to 17° C., and was then prepared as an O/W emulsion, using aturbine type homomixer at 6,500 rpm. This O/W emulsion was stirred atroom temperature for 3 hours to volatilize off the dichloromethane andsolidify the oil phase, which was then collected via centrifugation at1,500 rpm using a centrifuge (O5PR-22, Hitachi Limited). The oil phasewas again dispersed in distilled water, followed by centrifugation towash off the free drug etc. After the collected microcapsules were againdispersed in a small amount of distilled water, 50 mg of D-mannitol wasadded; the resulting dispersion was lyophilized to yield a powder. Thehuman interferon alpha activity of the microcapsules was 2.24×10⁶ IU/mgmicrocapsule.

EXPERIMENTAL EXAMPLE 1

About 14 mg of the microcapsules obtained in Example 6 (1.35 mg ofbiologically active peptide A contained) was dispersed in 0.5 ml of adispersant (a solution of 2.5 mg of carboxymethyl cellulose, 0.5 mg ofPolysorbate 80 and 25 mg of mannitol in distilled water). Thisdispersion was subcutaneously administered via 22-gauge injection needleto the backs of male SD rats at 10 weeks of age. Followingadministration, blood was regularly taken from each rat via the tailvein, and assayed for biologically active peptide A content by RIA. Theresults are given in Table 1.

                  TABLE 1                                                         ______________________________________                                        biologically active Peptide A Concentration                                   (ng/ml)                                                                                  Week    Week    Week  Week                                         Day 1      1       2       3     4     Week 6                                 ______________________________________                                        Experi-                                                                              1.75    4.41    4.31  3.19  2.43  0.76                                 mental                                                                        Example                                                                       ______________________________________                                    

When polylactic acid having a methyl-esterified terminal carboxyl groupwas used, low blood drug levels were obtained 1 day afteradministration, demonstrating very low initial drug release afteradministration. The blood drug level remained almost constant over a1-month period, indicating a good sustained-release property.

EXPERIMENTAL EXAMPLE 2

About 87 mg of the microcapsules obtained in Example 7 (5.0×10⁷ IU humaninterferon alpha contained) was dispersed in 0.5 ml of a dispersant (asolution of 2.5 mg of carboxymethyl cellulose, 0.5 mg of Polysorbate 80and 25 mg of mannitol in distilled water). This dispersion wassubcutaneously administered via 22-gauge injection needle to the backsof male SD rats at 8 weeks of age. Following administration, blood wasregularly taken from each rat via the tail vein, and assayed for humaninterferon alpha content by EIA. The results are given in Table 2.

EXPERIMENTAL EXAMPLE 3

The microcapsules obtained in Comparative Example 1 were treated in thesame manner as in Experimental Example 2 to determine the blood humaninterferon alpha content. The results are given in Table 2.

                  TABLE 2                                                         ______________________________________                                                Human Interferon Alpha Concentration (IU/ml)                                  3     6       Day    Day  Day  Day  Day                                       Hours Hours   1      2    3    7    9                                 ______________________________________                                        Experimental                                                                            5042.6  3934.3  162.3                                                                              122.6                                                                              139.9                                                                              113.8                                                                              43.8                            Example 2                                                                     Experimental                                                                            5127.2  6121.6   65.9                                                                               42.9                                                                               32.3                                                                               1.9  0.0                            Example 3                                                                     ______________________________________                                    

When a lactic acid/glycolic acid copolymer having an ethylated terminalcarboxyl group was used, initial drug release was suppressed, while ahigh stationary blood drug level was maintained, demonstrating a goodsustained-release property.

EXPERIMENTAL EXAMPLE 4

About 20 mg of the microcapsules obtained in Example 8 (5.0×10⁷ IU humaninterferon alpha contained) was dispersed in 0.5 ml of a dispersant (asolution of 2.5 mg of carboxymethyl cellulose, 0.5 mg of Polysorbate 80and 25 mg of mannitol in distilled water). This dispersion wassubcutaneously administered via 22-gauge injection needle to the backsof male SD rats at 8 weeks of age. Following administration, blood wasregularly taken from each rat via the tail vein, and assayed for humaninterferon alpha content by EIA.

Despite the fact that the dose per rat was almost the same as inExperimental Example 2, the blood human interferon alpha concentrationremained as high as 133 IU/ml even at day 14 following administration.Good sustained-release was obtained when a lactic acid/glycolic acidcopolymer having an ethylated terminal carboxyl group was used.

The ester of the present invention, can be used as a matrix forsustained-release preparation. The matrix is stable to light, heat,moisture, coloring etc., and is of low toxicity.

The sustained-release preparation produced by using the ester of thepresent invention offers stable drug release over an extended period oftime, ensuring sustained stable effect. Furthermore, thesustained-release preparation does not show excess drug release justafter administration.

What is claimed is:
 1. An ester formed at a terminal carboxyl group of astraight-chain polyester consisting essentially of anα-hydroxymonocarboxylic acid, the polyester having a weight-averagemolecular weight of about 1,500 to about 50,000.
 2. The ester accordingto claim 1, which is an ester formed at a terminal carboxyl group of alactic acid/glycolic acid copolymer.
 3. The ester according to claim 1,which is an alkyl ester.
 4. The ester according to claim 3, which is aC₁₋₃ alkyl ester.